Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) β-subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH

Abstract

The region between the 10th and 12th cysteine (Cys88-Cys105 in human thyroid-stimulating hormone β-subunit (hTSHβ)) of the glycoprotein hormone β-subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common α-subunit and has been implicated in regulating specificity between human choriogonadotropin (hCG) and human follicle- stimulating hormone (hFSH), but determinants of hTSH specificity are unknown. To characterize the role of this region for hTSH, we constructed hTSH chimeras in which the entire seatbelt region Cys88-Cys105 or individual intercysteine segments Cys88-Cys95 and Cys95-Cys105 were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys95-Cys105 segment of the seat-belt was more important for TSH receptor binding and signal transduction than the Cys88-Cys95 determinant loop region. Replacing the entire seat-belt of hTSHβ with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSH receptor binding and activation were abolished. Conversely, introduction of the hTSHβ seat-belt sequence into hCGβ generated an hCG chimera that bound to and activated the TSH receptor but not the CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH seat-belt residues did not possess any follitropic activity, and its thyrotropic activity was only slightly reduced. This may in part be due to the fact that the net charge of the seat-belt is similar in hTSH and hFSH but different from hCG. However, exchanging other regions of charge heterogeneity between hTSHβ and hFSHβ did not confer follitropic activity to hTSH. Thus, exchanging the seatbelt region between hTSH and hCG switches hormonal specificity in a mutually exclusive fashion. In contrast, the seat-belt appears not to discriminate between the TSH and the FSH receptors, indicating for the first time that domains outside the seat-belt region contribute to glycoprotein hormone specificity.